The kynurenic acid analog SZR104 induces cytomorphological changes associated with the anti-inflammatory phenotype in cultured microglia.

We previously showed the anti-inflammatory effects of kynurenic acid (KYNA) and its brain-penetrable analog N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-hydroxyquinoline-2-carboxamide (SZR104) both in vivo and in vitro. Here, we identified the cytomorphological effects of KYNA and SZR104 in secondary microglial cultures established from newborn rat forebrains. We quantitatively analyzed selected morphological aspects of microglia in control (unchallenged), lipopolysaccharide (LPS)-treated (challenged), KYNA- or SZR104-treated, and LPS + KYNA or LPS + SZR104-treated cultures. Multicolor immunofluorescence labeling followed by morphometric analysis (area, perimeter, transformation index, lacunarity, density, span ratio, maximum span across the convex hull, hull circularity, hull area, hull perimeter, max/min radii, mean radius, diameter of bounding circle, fractal dimension, roughness, circularity) on binary (digital) silhouettes of the microglia revealed their morphological plasticity under experimental conditions. SZR104 and, to a lesser degree, KYNA inhibited proinflammatory phenotypic changes. For example, SZR104 treatment resulted in hypertrophied microglia characterized by a swollen cell body, enlarged perimeter, increased transformation index/decreased circularity, increased convex hull values (area, perimeter, mean radius, maximum span, diameter of the bounding circle and hull circularity), altered box-counting parameters (such as fractal dimension), and increased roughness/decreased density. Taken together, analysis of cytomorphological features could contribute to the characterization of the anti-inflammatory activity of SZR104 on cultured microglia.

Kynurenic Acid and Its Analog SZR104 Exhibit Strong Antiinflammatory Effects and Alter the Intracellular Distribution and Methylation Patterns of H3 Histones in Immunochallenged Microglia-Enriched Cultures of Newborn Rat Brains.

Kynurenic acid (KYNA) is implicated in antiinflammatory processes in the brain through several cellular and molecular targets, among which microglia-related mechanisms are of paramount importance. In this study, we describe the effects of KYNA and one of its analogs, the brain-penetrable SZR104 (N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-hydroxyquinoline-2-carboxamide), on the intracellular distribution and methylation patterns of histone H3 in immunochallenged microglia cultures. Microglia-enriched secondary cultures made from newborn rat forebrains were immunochallenged with lipopolysaccharide (LPS). The protein levels of selected inflammatory markers C-X-C motif chemokine ligand 10 (CXCL10) and C-C motif chemokine receptor 1 (CCR1), histone H3, and posttranslational modifications of histone H3 lys methylation sites (H3K9me3 and H3K36me2, marks typically associated with opposite effects on gene expression) were analyzed using quantitative fluorescent immunocytochemistry and western blots in control or LPS-treated cultures with or without KYNA or SZR104. KYNA and SZR104 reduced levels of the inflammatory marker proteins CXCL10 and CCR1 after LPS-treatment. Moreover, KYNA and SZR104 favorably affected histone methylation patterns as H3K9me3 and H3K36me2 immunoreactivities, and histone H3 protein levels returned toward control values after LPS treatment. The cytoplasmic translocation of H3K9me3 from the nucleus indicated inflammatory distress, a process that could be inhibited by KYNA and SZR104. Thus, KYNA signaling and metabolism, and especially brain-penetrable KYNA analogs such as SZR104, could be key targets in the pathway that connects chromatin structure and epigenetic mechanisms with functional consequences that affect neuroinflammation and perhaps neurodegeneration.

Hippocampal Sclerosis in Pilocarpine Epilepsy: Survival of Peptide-Containing Neurons and Learning and Memory Disturbances in the Adult NMRI Strain Mouse.

The present experiments reveal the alterations of the hippocampal neuronal populations in chronic epilepsy. The mice were injected with a single dose of pilocarpine. They had status epilepticus and spontaneously recurrent motor seizures. Three months after pilocarpine treatment, the animals were investigated with the Barnes maze to determine their learning and memory capabilities. Their hippocampi were analyzed 2 weeks later (at 3.5 months) with standard immunohistochemical methods and cell counting. Every animal displayed hippocampal sclerosis. The neuronal loss was evaluated with neuronal-N immunostaining, and the activation of the microglia was measured with Iba1 immunohistochemistry. The neuropeptide Y, parvalbumin, and calretinin immunoreactive structures were qualitatively and quantitatively analyzed in the hippocampal formation. The results were compared statistically to the results of the control mice. We detected neuronal loss and strongly activated microglia populations. Neuropeptide Y was significantly upregulated in the sprouting axons. The number of parvalbumin- and calretinin-containing interneurons decreased significantly in the Ammon's horn and dentate gyrus. The epileptic animals displayed significantly worse learning and memory functions. We concluded that degeneration of the principal neurons, a numerical decrease of PV-containing GABAergic neurons, and strong peptidergic axonal sprouting were responsible for the loss of the hippocampal learning and memory functions.

Sensitivity of Rodent Microglia to Kynurenines in Models of Epilepsy and Inflammation In Vivo and In Vitro: Microglia Activation is Inhibited by Kynurenic Acid and the Synthetic Analogue SZR104.

Kynurenic acid is an endogenous modulator of ionotropic glutamate receptors and a suppressor of the immune system. Since glutamate and microglia are important in the pathogenesis of epilepsy, we investigated the possible action of the synthetic kynurenic acid analogue, SZR104, in epileptic mice and the action of kynurenic acid and SZR104 on the phagocytotic activity of cultured microglia cells. Pilocarpine epilepsy was used to test the effects of SZR104 on morphological microglia transformation, as evaluated through ionized calcium-binding adaptor molecule 1 (Iba1) immunohistochemistry. Microglia-enriched rat secondary cultures were used to investigate phagocytosis of fluorescent microbeads and Iba1 protein synthesis in control and lipopolysaccharide-challenged cultures. SZR104 inhibited microglia transformation following status epilepticus. Kynurenic acid and SZR104 inhibited lipopolysaccharide-stimulated phagocytotic activity of microglia cells. Although kynurenic acid and its analogues proved to be glutamate receptor antagonists, their immunosuppressive action was dominant in epilepsy. The inhibition of phagocytosis in vitro raised the possibility of the inhibition of genes encoding inflammatory cytokines in microglial cells.

Differential expression of the c-fos protein and synaptophysin in zebrin II positive and zebrin II negative cerebellar cortical areas in 4-aminopyridine seizures.

The present study examined temporal activation patterns of rat cerebellar cortical neurons in 4-aminopyridine induced seizures, using c-fos protein as a marker of neuronal activity. C-fos-containing cells were counted in each cerebellar cortical layer, and cell count was compared between zebrin II positive and zebrin II negative bands of the lobules of the vermis and cerebellar hemispheres. We found significant activation of granule cells and interneurons of the molecular layer in zebrin II positive bands. The Purkinje cells, in contrast, exhibited non-significant, scattered c-fos immunoreactivity across all bands. Fluctuation of synaptophysin expression in the mossy fibre rosettes of the granular layer was determined via light microscopic immunohistochemistry. We detected a transient, significant decrease in synaptophysin staining density following 4-aminopyridine seizures, which may indicate short-term synaptic depression. We also identified different timing of increased c-fos expression in the neurons of the cerebellar cortex in different cortical zones. In particular, the activation pattern of the interneurons of the molecular layer reflected the climbing fibre distribution, reflecting the zonal olivo-cortico-nuclear organization. Seizure-induced activation of the granule cells corresponded with the zebrin II positive zones. This observation raises the possibility that zebrin II positive compartments may be more susceptible to cerebellar convulsions.

The Reactive Plasticity of Hippocampal Ionotropic Glutamate Receptors in Animal Epilepsies.

Ionotropic glutamate receptors (iGluRs) mediate the synaptic and metabolic actions of glutamate. These iGluRs are classified within the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type, kainate-type, and N-methyl-d-aspartate (NMDA)-type functional receptor families. The iGluR assemblies are regulated by transcription, alternative splicing, and cytoplasmic post-translational modifications. The iGluR subunit proteins are transported from the endoplasmic reticulum, inserted into the synaptic membranes, and anchored at their action site by different scaffolding and interacting proteins. The functional properties of iGluRs depend on their subunit composition, the amino acid sequence of the protein domains, and the scaffolding proteins in the synaptic membranes. The iGluRs are removed from the membranes by enzymatic action and endocytosis. Hippocampal iGluRs are rearranged through the upregulation and downregulation of the subunits following deafferentation and epileptic seizures. The rearrangement of iGluRs and the alteration of their subunit composition transform neurons into "pathological" cells, determining the further plasticity or pathology of the hippocampal formation. In the present review, we summarize the expression of AMPA, kainate, and NMDA receptor subunits following deafferentation, repeated mild seizures, and status epilepticus. We compare our results to literature descriptions, and draw conclusions as to the reactive plasticity of iGluRs in the hippocampus.

Numerical and Theoretical Aspects of the DMRG-TCC Method Exemplified by the Nitrogen Dimer.

In this article, we investigate the numerical and theoretical aspects of the coupled-cluster method tailored by matrix-product states. We investigate formal properties of the used method, such as energy size consistency and the equivalence of linked and unlinked formulation. The existing mathematical analysis is here elaborated in a quantum chemical framework. In particular, we highlight the use of what we have defined as a complete active space-external space gap describing the basis splitting between the complete active space and the external part generalizing the concept of a HOMO-LUMO gap. Furthermore, the behavior of the energy error for an optimal basis splitting, i.e., an active space choice minimizing the density matrix renormalization group-tailored coupled-cluster singles doubles error, is discussed. We show numerical investigations on the robustness with respect to the bond dimensions of the single orbital entropy and the mutual information, which are quantities that are used to choose a complete active space. Moreover, the dependence of the ground-state energy error on the complete active space has been analyzed numerically in order to find an optimal split between the complete active space and external space by minimizing the density matrix renormalization group-tailored coupled-cluster error.

Non-competitive antagonists of NMDA and AMPA receptors decrease seizure-induced c-fos protein expression in the cerebellum and protect against seizure symptoms in adult rats.

The aim of the present study was to examine the role of ionotropic glutamate receptors in the cerebellum during generalized seizures. Epileptic neuronal activation was evaluated through the immunohistochemical detection of c-fos protein in the cerebellar cortex. Generalized seizures were precipitated by the intraperitoneal injection of 4-aminopyridine. The animals were pretreated with the NMDA receptor antagonists MK-801 (2 mg/kg), amantadine (50 mg/kg), and the AMPA receptor antagonist GYKI 52466 hydrochloride (50 mg/kg). Two hours after 4-aminopyridine injection, the number of c-fos immunostained cell nuclei was counted in serial immunohistochemical sections of the cerebellar vermis. The number of c-fos immunostained cell nuclei in the granular layer decreased significantly in animals pretreated with the glutamate receptor antagonists compared to the untreated animals having convulsion. We can conclude that mossy fiber stimulation exerts its seizure-generating action mainly through the ionotropic glutamate receptors of the mossy fiber synapses. Both NMDA and AMPA receptor antagonists are effective in reducing glutamate-mediated postsynaptic effects in the cerebellar cortex.

Comparative immunohistochemical study of the effects of pilocarpine on the mossy cells, mossy fibres and inhibitory neurones in murine dentate gyrus.

Treatment with pilocarpine (PILO) induces variable degrees of loss of mossy cells (MCs) and mossy fibre (MF) sprouting in rodents, the relationships of which have not been examined in individual animals. Our aim was to test whether the loss of MCs and MF sprouting are coupled processes in PILO-treated rodents. Animals which exhibited intense PILO-induced convulsions for at least 30 min were used in this study. After a 2-month survival period, the incidence of epileptic seizures was checked individually by neuropeptide-Y (NPY) immunohistochemistry, and the numbers of MCs were counted by means of immunohistochemistry, for calretinin (CR) in mice and calcitonin gene-related peptide (CGRP) in rats. MF sprouting was checked by using Timm's silver-sulphide method for zinc. In our comparative studies, NPY immunohistochemistry resulted in more positive animals than on zinc staining. The CR immunoreactivity remained unchanged even in those mice that displayed MF sprouting and greatly increased NPY immunoreactivity. CR immunoreactivity was also verified after transection of the fornix to exclude the extrahippocampal source of this peptide. However, the CGRP immunoreactivity was severely reduced in those rats that exhibited simultaneous increases in zinc content and NPY immunoreactivity in the supragranular layer and stratum lucidum. Our findings suggest that the MCs survive PILO treatment in mice, but not in rats. There is direct evidence of a close relationship between the loss of MCs and MF sprouting in rats, but not in mice. Thus, similar PILO seizures may result from different changes in the neuronal circuits of rodents.

Interstrain differences of ionotropic glutamate receptor subunits in the hippocampus and induction of hippocampal sclerosis with pilocarpine in mice.

Rodent strains used in epilepsy research have various neurological characteristics. These differences were suggested to be attributed to the diverse densities of the ionotropic glutamate receptor (iGluR) subunits. However, previous studies failed to find interstrain differences in the hippocampal receptor levels. We supposed that a detailed layer-to-layer analysis of the iGluR subunits in the hippocampus might reveal strain-dependent differences in their base lines and reactions induced by pilocarpine (PILO) between two mouse strains without documented ancestors. Levels of iGluR subunits in Balb/c and NMRI mice were compared using semiquantitative immunohistochemistry. The alterations in the neuronal circuitry were validated by neuropeptide Y (NPY) and neuronal nuclear antigen (NeuN) immunostainings. Immunohistochemistry showed interstrain laminar differences in some subunits of both the control and PILO-treated animals. The seizure-induced irreversible neuronal changes were accompanied by reduced GluA1 and GluA2 levels. Their changes were inversely correlated in the individual NMRI mice by Pearson's method. Increase in NPY immunoreactivity showed positive correlation with GluA1, and negative correlation with GluA2. The NMRI strain was susceptible to PILO-induced hippocampal sclerosis, while the Balb/c animals showed resistance. Basal levels of iGluRs differ in mouse strains, which may account for the interstrain differences in their reactions to the convulsant.

Repeated application of 4-aminopyridine provoke an increase in entorhinal cortex excitability and rearrange AMPA and kainate receptors.

Entorhinal cortex is a highly epilepsy-prone brain region. Effects of repetitive seizures on ionotropic glutamate receptors (iGluRs) were investigated in rat entorhinal cortex slices. Seizures were induced by daily administration of 4-aminopyridine (4-AP). Electrophysiological, pharmacological and histological investigations were carried out to determine changes in synaptic efficacy and in sensitivity of iGluRs due to recurring seizures. Repeated 4-AP-induced seizures increased the amplitude of evoked synaptic field responses in rat entorhinal cortical slices. While vulnerability to inhibition of AMPA receptors by the specific antagonist GYKI 52466 was slightly reduced, responsiveness to NMDA receptor antagonist APV remained unaffected. Testing of bivalent cation permeability of iGluRs revealed reduced Ca(2+)-influx through non-NMDA receptors. According to the semi-quantitative histoblot analysis GluA1-4, GluA1, GluA2, GluK5, GluN1 and GluN2A subunit protein expression differently altered. While there was a marked decrease in the level of GluA1-4, GluA2 and GluK5 receptor subunits, GluA1 and GluN2A protein levels moderately increased. The results indicate that brief convulsions, repeated daily for 10 days can increase overall entorhinal cortex excitability despite a reduction in AMPA/kainate receptor activity, probably through the alteration of local network susceptibility.

Immunohistochemistry of cerebellar seizures: mossy fiber afferents play an important role in seizure spread and initiation in the rat.

Clinical reports suggest the participation of the cerebellum in epilepsy. Mossy fibers are the main excitatory afferents of the cerebellar cortex; most of them use glutamate and strongly excite granule cells through NMDA and AMPA receptors. The role of the ponto-cerebellar mossy fibers in cerebellar neuronal hyperactivity was investigated in the present study in experimental adult Wistar rats. We detected neuronal hyperactivity through the expression of the glutamate-induced c-fos protein, by means of immunohistochemistry and immunoblotting in the vermis and in the hemispheres. Generalized seizures were induced by means of intraperitoneal 4-aminopyridine injections. Following the 4-aminopyridine seizures, the c-fos expression of cerebellar granule cells was significantly elevated at 1.5h in every lobule. Maximum c-fos expression was seen at 3h. The role of the ponto-cerebellar mossy fiber afferents in the induction of c-fos expression was examined after the transection of the middle cerebellar peduncle on the left side. Immunohistochemical analysis 14 days after the surgery revealed that the synapsin I immunoreactivity was significantly reduced in the cerebellar cortex on the operated side, compared to the sham-operated controls and to the non-operated cerebellar hemisphere of the operated animals, indicating the degeneration of mossy fiber terminals. Transection of the middle cerebellar peduncle suppressed cerebellar c-fos expression in the vermis and in the hemispheres significantly. These findings suggest the strong involvement of the middle cerebellar peduncle and the ponto-cerebellar mossy fibers in the pathophysiology of cerebellar epilepsy.

New silver-gold intensification method of diaminobenzidine for double-labeling immunoelectron microscopy.

The available methods for double-labeling preembedding immunoelectron microscopy are highly limited because not only should the ultrastructure be preserved, but also the different antigens should be visualized by reaction end products that can be clearly distinguished in gray-scale images. In these procedures, one antigen is detected with 3,3'-diaminobenzidine (DAB) chromogen, resulting in a homogeneous deposit, whereas the other is labeled with either a gold-tagged immunoreagent, or DAB polymer, on the surface of which metallic silver is precipitated. The detection of the second antigen is usually impeded by the first, leading to false-negative results. The authors aimed to diminish this hindrance by a new silver intensification technique of DAB polymer, which converts the deposit from amorphous to granular. The method includes three major postdevelopmental steps: (1) treatment of nickel-enhanced DAB with sulfide, (2) silver deposition in the presence of hydroquinone under acidic conditions, and (3) precious metal replacement with gold thiocyanate. This new sulfide-silver-gold intensification of DAB (SSGI) allows a subsequent detection of other antigens using DAB. In conclusion, the new technique loads fine gold particles onto the DAB deposit at a very low background level, thereby allowing a reliable discernment between the elements stained for the two antigens at the ultrastructural level.

Comparative immunohistochemistry of synaptic markers in the rodent hippocampus in pilocarpine epilepsy.

Pilocarpine-induced epileptic state (Status epilepticus) generates an aberrant sprouting of hippocampal mossy fibers, which alter the intrahippocampal circuits. The mechanisms of the synaptic plasticity remain to be determined. In our studies in mice and rats, pilocarpine-induced seizures were done in order to gain information on the process of synaptogenesis. After a 2-month survival period, changes in the levels of synaptic markers (GAP-43 and Syn-I) were examined in the hippocampus by means of semi-quantitative immunohistochemistry. Mossy fiber sprouting (MFS) was examined in each brain using Timm's sulphide-silver method. Despite the marked behavioral manifestations caused by pilocarpine treatment, only 40% of the rats and 56% of the mice showed MFS. Pilocarpine treatment significantly reduced the GAP-43 immunoreactivity in the inner molecular layer in both species, with some minor differences in the staining pattern. Syn-I immunohistochemistry revealed species differences in the sprouting process. The strong immunoreactive band of the inner molecular layer in rats corresponded to the Timm-positive ectopic mossy fibers. The staining intensity in this layer, representing the ectopic mossy fibers, was weak in the mouse. The Syn-I immunoreactivity decreased significantly in the hilum, where Timm's method also demonstrated enhanced sprouting. This proved that, while sprouted axons displayed strong Syn-I staining in rats, ectopic mossy fibers in mice did not express this synaptic marker. The species variability in the expression of synaptic markers in sprouted axons following pilocarpine treatment indicated different synaptic mechanisms of epileptogenesis.

Antinociceptive effect of vinpocetine--a comprehensive survey.

Blockade of retrograde transport of nerve growth factor (NGF) in a peripheral sensory nerve is known to induce transganglionic degenerative atrophy (TDA) of central sensory terminals in the upper dorsal horn of the related, ipsilateral segments(s) of the spinal cord. The ensuing temporary blockade of transmission of nociceptive impulses has been utilized in the therapy of intractable pain, using transcutaneous iontophoresis of the microtubule inhibitors vincristin and vinblastin, drugs which inhibit retrograde transport of NGF. Since microtubule inhibition might inhibit (at least theoretically) mitotic processes in general, we sought to find a drug which inhibits retrograde transport of NGF without microtubule inhibition. Vinpocetine, a derivate of vincamine, which does not interfere with microtubular function, was found to inhibit retrograde axoplasmic transport of NGF in peripheral sensory nerves, similarly to vincristin and vinblastin. Blockade of NGF transport is followed by transganglionic degenerative atrophy in the segmentally related, ipsilateral superficial spinal dorsal horn, characterized by depletion of the marker enzymes of nociception, fluoride resistant acid phosphatase (FRAP) and thiamine monophosphatase (TMP) from the Rolando substance and by decrease of the pain-related neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from lamina I-II-III. Based upon these findings, it has been suggested that vinpocetine may result in a locally restricted decrease of nociception. Herewith, the structural and behavioral effects of perineurally administered vinpocetine are discussed. Nociception, induced by intraplantar injection of formalin, was mitigated by perineural application of vinpocetine; also formalin-induced expression of c-fos in the ipsilateral, segmentally related superficial dorsal horn, was prevented by this treatment. Since vinpocetine is not a microtubule inhibitor, its mode of action is enigmatic. It is assumed that the effect of vinpocetine might be related to interaction with membrane-trafficking proteins, such as signalling endosomes and the endocytosis-mediating "pincher" protein, involved in retrograde axoplasmic transport of NGF, or to interaction with glial elements, recently reported to be involved in the modulation of pain in the spinal cord. Based on animal experiments it is assumed that the temporary, locally restricted decrease of nociception, induced by vinpocetine applied via transcutaneous iontophoresis, might open up new avenues in the clinical treatment of intractable pain.

Single-dose and chronic corticosterone treatment alters c-Fos or FosB immunoreactivity in the rat cerebral cortex.

The aim of this study was to examine the effects of single-dose and chronic corticosterone treatment on the inducible transcription factor c-Fos and FosB, and thereby to estimate the effects of high-doses of corticosterone on calcium-dependent neuronal responses in the rat cerebral cortex. At the same time we investigated the distribution of interneurons containing calretinin (CR), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) in chronically treated animals in order to collect data on the involvement of inhibitory neurons in this process. Adult male rats were injected subcutaneously with 10mg corticosterone, whereas controls received the vehicle (sesame oil). The animals were fixed by transcardial perfusion 12 and 24h following single corticosterone injection, and the brains were processed for c-Fos and FosB immunohistochemistry. To investigate the effects of repeated corticosterone administration, rats were daily treated with the same amount of corticosterone (10mg/animal, subcutaneously) for 21 days. Controls were injected with vehicle. At the end of the experiment, the rats were perfused and immunohistochemistry was used to detect the presence of the FosB protein, CR, VIP and NPY. Quantitative evaluation of immunolabelled cells was performed in the neocortex and the hippocampus. The number of immunoreactive nuclei per unit area was used as a quantitative measure of the effects of corticosterone. It was found that a single-dose administration of corticosterone resulted in a significant, time-dependent increase of c-Fos protein immunoreactivity in the granule cell layer of the dentate gyrus, as well as in regions CA1 and CA3 of the hippocampus 12 and 24h post-injection with respect to control animals. Significant enhancement of c-Fos immunoreactivity was also observed in the neocortex at 12 and 24h post-injection. Single-dose treatment did not significantly alter FosB immunolabelling. Repeated administration of corticosterone produced a complex pattern of changes in FosB immunolabelling: significant increase in FosB immunoreactivity was detected in the granule cell layer of the dentate gyrus, with no significant changes in the CA1 and CA3 layers of the hippocampus and in the neocortex. However, a significant decrease of FosB induction in the neocortex was observed in chronically treated rats in comparison to single-dose injected animals (12h before immunohistochemistry). Analysis of immunohistochemical detection of interneuronal markers revealed a significant reduction of the CR immunolabelling in the CA3 area of the hippocampus. No changes in VIP or NPY immunoreactivity were found in the Ammon's horn 3 weeks following daily corticosterone treatment. NPY immunoreactivity was significantly attenuated in the neocortex. The present data suggest that single-dose corticosterone treatment increases immunoreactivity of c-Fos protein in a time-dependent manner, 12 and 24h post-injection in the rat hippocampus and the neocortex, whereas chronic corticosterone treatment influences FosB immunoreactivity, primarily in the dentate gyrus. Chronic corticosterone administration seems to affect CR levels in the CA3 area of the hippocampus.

Repeated 4-aminopyridine induced seizures diminish the efficacy of glutamatergic transmission in the neocortex.

Systemic administration of the potassium channel blocker 4-aminopyridine (4-AP) elicits acute convulsions. Synchronized tonic-clonic activity develops during the first hour after the treatment. However, subsequent chronic spontaneous seizures do not appear which suggests changes in neuronal excitability. The aim of our present work was to evaluate alterations in the glutamatergic transmission in the somatosensory cortex of rats following daily, brief convulsions elicited by 4-AP treatment. Changes in general neuronal excitability and pharmacological sensitivity of glutamate receptors were tested in ex vivo electrophysiological experiments on brain slices. In parallel studies quantitative changes in subunit composition of glutamate receptors were determined with immunohistoblot technique, together with the analysis of kainate induced Co2+ uptake. The results of our coordinated electrophysiological, receptor-pharmacological and histoblot studies demonstrated that repeated, daily, short convulsions resulted in a significant decrease of the general excitability of the somatosensory cortex together with changes in ionotropic glutamate receptor subunits. The relative inhibitory effect of the AMPA receptor antagonist, however, did not change. The NMDA receptor antagonist exerted somewhat stronger effect in the slices from convulsing animals. 4-AP pretreatment resulted in the attenuation of kainate induced Co2+ uptake, which suggests either reduction in non-NMDA receptors numbers or reduction in their Ca2+ permeability. Repeated seizures decreased GluR1-4 AMPA receptor subunit levels in all cortical layers with a relaitve increase in GluR1 subunits. While the principle NR1 NMDA receptor subunit showed no significant change, the staining density of NR2A subunit increased. These changes in ionotropic glutamate receptors are consistent with reduced excitability at glutamatergic synapses following repeated 4-AP induced seizures.

Late expression of FosB transcription factor in 4-aminopyridine-induced seizures in the rat cerebral cortex.

In this study, the immunolocalization of FosB transcription factor was investigated in acute and chronic experimental models of seizures induced by 4-aminopyridine. Wistar rats were injected intraperitoneally daily with 5mg/kg 4-aminopyridine for 1, 4, 8 and 12 days and sacrificed 24h after the last injection. Corresponding control groups received the solvent of 4-aminopyridine. Immunohistochemistry revealed an increase in FosB immunolabelling in the frontal cortex in 4-aminopyridine-treated animals compared to controls, both in acute and chronic time course groups. The dentate gyrus displayed elevated FosB immunopositivity only after repeatedly applied convulsant (4-aminopyridine), i.e. following 4, 8 and 12 days of treatment, but no significant immunolocalization was observed in the hippocampus proper. The neuronal localization of FosB after 12 days of 4-aminopyridine-induced convulsions was analysed by means of FosB-parvalbumin double immunolabelling. The increased number of double-labelled cells was significant in the frontal cortex, hilum of the dentate fascia and region CA1 of the hippocampus. We conclude that the studied neocortical and allocortical areas showed a different pattern of FosB immunolocalization, which suggests a relative deficiency of transcriptional regulation in the Ammon's horn and may be responsible for distinct response to seizure-induced cellular insult.

Mitigation of nociception via transganglionic degenerative atrophy: possible mechanism of vinpocetine-induced blockade of retrograde axoplasmic transport.

Vinpocetine, a derivative of vincamine, widely used in the clinical pharmacotherapy of cerebral circulatory diseases, inhibits retrograde axoplasmic transport of nerve growth factor (NGF) in the peripheral nerve, resulting in transganglionic degenerative atrophy (TDA) in the related ipsilateral superficial spinal dorsal horn, as shown in our previous publications. TDA induced by vinpocetine has been demonstrated to be followed by depletion of the marker enzyme fluoride-resistant acid phosphatase (FRAP) and its isoenzyme thiamine monophosphatase (TMP), and by the decrease in the pain-related neuropeptide substance P from laminae I-II-(III) from the segmentally related, ipsilateral substance of Rolando of the spinal cord. In the present paper, we report on the behavioral effects of perineurally administered vinpocetine. Nociception, induced by intraplantar injection of formalin, was mitigated by vinpocetine; increased expression of c-fos in the ipsilateral, segmentally related upper dorsal horn was also prevented. Since vinpocetine is not a microtubule inhibitor, and its chemical structure differs from that of vincristin and vinblastin (used formerly by us in the therapy of intractable, chronic neuropathic pain), its mode of action is enigmatic. We assume that the effect of vinpocetine in blocking retrograde axoplasmic transport of NGF might be related to its interaction with membrane trafficking proteins, such as signalling endosomes and the endocytosis-mediating "pincher" protein. Temporary, locally restricted decrease of nociception, induced by vinpocetine, might be useful in the clinical treatment of intractable, chronic neuropathic pain, since vinpocetine can successfully be applied by transcutaneous iontophoresis.

Blockade of AMPA-receptors attenuates 4-aminopyridine seizures, decreases the activation of inhibitory neurons but is ineffective against seizure-related astrocytic swelling.

The neurotransmitter glutamate plays a pivotal role in the development of the neuropathological sequelae following acute seizures. Our previous data proved the efficacy of the NMDA-receptor antagonists on the symptoms, survival and neuronal activation in the 4-aminopyridine- (4-AP) induced seizures. In this study, we examined the effects of two different doses of a non-competitive, selective, allosteric AMPA-receptor antagonist, GYKI 52466. GYKI 52466 was effective in prolonging the latency to generalised seizures and reduction of seizure mortality. However, the effects on neuronal c-fos expression and astrocyte swelling were complex. The 25mg/kg dose of GYKI 52466 was effective in reducing the c-fos immunoreactivity (IR) in the hippocampus only. In the neocortex the overall c-fos-IR cell counts were increased significantly. Investigation of the neocortical parvalbumin-containing interneuron population proved that GYKI 52466 decreased c-fos expression. The 50mg/kg dose of GYKI 52466 significantly reduced the c-fos-IR in the neo- and allocortex, not only in principal neurons, but also in the parvalbumin-positive interneurons. The GYKI 52466-pretreatment did not prevent the astrocyte swelling in the investigated cortical areas; thus we conclude that the AMPA-receptors have little if any involvement in the in the mediation of neuropathological alterations in acute convulsions.